Author Proof A American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 9999:1–4 (2004) Association Analysis of NOTCH 4 Polymorphisms With Schizophrenia Among Two Independent Family Based Samples S. Prasad, 1 K.V. Chowdari, 2 J. Wood, 2 T. Bhatia, 1 S.N. Deshpande, 3 V.L. Nimgaonkar, 2 and B.K. Thelma 1 * 1 Department of Genetics, University of Delhi, South Campus, Benito Juarez Road, New Delhi, India 2 Department of Psychiatry and Human Genetics, University of Pittsburgh School of Medicine and Graduate School of Public Health, Pittsburgh, Pennsylvania 3 Department of Psychiatry, Dr. Ram Manohar Lohia Hospital, New Delhi, India The present study investigated polymorphisms of the NOTCH 4 gene in two independent samples from India and USA, consisting of patients with schizophrenia and their parents (n ¼ 182, and n ¼ 148 ‘trios,’ respectively). Five DNA markers, namely (GAAG) n , (TAA) n , SNP1, SNP2, and (CTG) n were evaluated. Transmission distortion, con- sistent with a modest association was detect- ed among both samples. Additional association studies at this locus are warranted. ß 2004 Wiley-Liss, Inc. KEY WORDS: Notch 4; schizophrenia; genetics; association; SNPs INTRODUCTION The etiology of schizophrenia is multi-factorial, with the possibility of several susceptibility genes conferring modest risk. Despite promising results, it has been difficult to identify genes that confer risks of a relatively small magnitude for common human diseases. Typically, promising associations have not been replicated in follow-up studies having sufficient power [Dahlman et al., 2002]. The initial reports tend to overestimate the effect size and artifacts can undoubtedly inflate the initial results [Cohen, 1999]. On the other hand, considerable inter-study heterogeneity contributes to difficul- ties with replications. Thus, meta-analyses may be required to resolve inconsistencies [Ioannidis et al., 2001]. Unfortunately, reluctance to publish non-significant associations may lead to premature loss of interest in a ‘true’ susceptibility gene. We illustrate this problem with respect to a controversial associa- tion between NOTCH 4 and schizophrenia. Linkage and association studies have been instrumental in the effort to map schizophrenia susceptibility genes. In particular, genome wide linkage scans have repeatedly identified 6p21-24 region as a putative susceptibility region [Antonarakis et al., 1995; Schwab et al., 1995; Straub et al., 1995; Wang et al., 1995; Turecki et al., 1997; Lindholm et al., 1999; Bailer et al., 2000; Schwab et al., 2000; Lewis et al., 2003]. A detailed account of studies in this region has been published recently by Skol et al. [2003]. The reported association of schizophrenia with NOTCH 4 [Wei and Hemmings, 2000] and Dysbindin (DTNBP1) genes [Straub et al., 2002], localized in this region are consistent with the linkage results. NOTCH family of genes are involved in inter-cellular signaling and encode trans-membrane receptors. Some human diseases have been found to be associated with NOTCH mutations; for example, leukemia and CADASIL (cerebral autosomal domi- nant arteriopathy with subcortical infarcts and leukoencepha- lopathy) [Joutel and Tournier-Lasserve, 1998] [reviewed by Kageyama and Ohtsuka, 1999]. Wei and Hemmings [2000] reported a significant association of schizophrenia with polymorphism at NOTCH 4 gene, located within the HLA class III region. The Transmission Disequili- brium Test (TDT), which detects association only in the pre- sence of linkage, revealed significant transmission distortion for three NOTCH 4 markers in this study. A subsequent large international consortium detected a trend for over-transmis- sion of 13 repeat allele of (TAA) n marker and 11 repeat allele of (CTG) n to schizophrenia patients in German-Israeli sib pair samples [Sklar et al., 2001]. Investigation of these markers in a large Chinese sample did not reveal any association [Fan et al., 2002]. In addition, four other case-control studies failed to detect significant association [Imai et al., 2001; McGinnis et al., 2001; Ujike et al., 2001; Wassink et al., 2003]. Thus, a re- sounding argument for absence of a genetic association could be made. Nonetheless, each of these studies evaluated only a fraction of all the identified polymorphisms at the NOTCH 4 gene, which spans 56.8 kb. Given the highly variable and un- certain linkage disequilibrium observed over small distances in the human genome, identification of the primary association conferring susceptibility could arguably be elusive [Martin et al., 2000]. Plausible functional correlations have recently been proposed for the putative risk allele(s). While it has long been known that NOTCH 4 is expressed in the human brain [Uyttendaele et al., 1996], allelic associations of a (CTG) n polymorphism with brain volumes estimated from MRI scans, as well as performance on the Wisconsin Card Sort Test (WCST) were recently reported among individuals with schizophrenia, as well as unaffected controls [Wassink et al., 2003]. These analyses suggest credible mechanisms of patho- genesis. With this background, we investigated associations at NOTCH 4 in two large and independent family based samples. MATERIALS AND METHODS Clinical Analysis Participants were recruited through ongoing genetic epi- demiological studies at Pittsburgh and New Delhi, using AJMB-03-223 Grant sponsor: XXX Q1 (to VLN, BKT, SND); Grant numbers: RO3 TW00730, N-443-645; Grant sponsor: XXX Q2 (to VLN); Grant numbers: MH 63450; MH56242; MH01489; Grant sponsor: XXX Q3 (to BKT, SND); Grant number: BT/PR2425/MED13/089/ 2001; Grant sponsor: Council of Scientific and Industrial Research, New Delhi (Senior Research Fellowship to SP). *Correspondence to: B.K. Thelma, Department of Genetics, University of Delhi, South Campus, Benito Juarez Road, New Delhi 110021, India. E-mail: humgen@vsnl.com Received 9 December 2003; Accepted 10 May 2004 DOI 10.1002/ajmg.b.30083 Published online 00 Month 2004 in Wiley InterScience (www.interscience.wiley.com) ß 2004 Wiley-Liss, Inc.